The polysaccharide chains of heparan sulfate proteoglycan (HSPG) may undergo marked variation in the relative amounts of N-acetylated and N-sulfated glucosamine (G1cN) residues, the relative amounts of D-glucuronic and L-iduronic acid residues, and the degree of sulfation. These polymers, found in all cells examined to date, are synthesized, secreted, endocytosed and catabolized rapidly in cultured hepatocytes and other cells; yet, their physiological role is unknown. Our long term objective is to determine the role(s) played by these structurally variable and rapidly turned over polymers. To these ends, the specific aims for the immediate future are to describe the metabolism of HSPG in a cultured hepatocyte cell line with particular emphasis on the intracellular routing of HSPG during synthesis, secretion, and endocytosis and the structural changes in HSPG as it passes from one organelle to another. Particular emphasis will be placed on the structural features of the heparan sulfate (HS) chains at different stages of HSPG metabolism. The structural analyses will involve HPLC profiling of the oligosaccharide fragments formed by nitrous acid cleavage of HS chains either at the N-sulfated G1cN residues, or at the N-acetylated G1cN residues after hydrazinolysis of the polymers. Several of the profiling procedures will be developed during the course of these studies and will be extensions of a previous profiling procedure developed for determining the structure for heparan.